Our overall objective is to understand retinal angiogenesis in a context of development and ocular disease, using mice as a model system. Retinal vascular diseases, including proliferative diabetic retinopathy and neovascular age related macular degeneration, are major causes of impaired vision and blindness. One of the central angiogenic signaling pathways in the retina is Norrin/Frizzled4 signaling. Norrin signals via its receptor Frizzled4 and the co-receptor LRP5 on vascular endothelial cells to activate ss-catenin signaling and transcriptional programs. The pathway plays a critical role in instructing retinal vascular morphogenesis and blood-retina barrier formation. Impaired Norrin/Frizzled4 signaling causes familial exudative vitreoretinopathy, an inherited disease that can cause blindness. The focus of this application is on the membrane protein TSPAN12, a novel and essential component of Norrin/Frizzled4 signaling. Tspan12 gene disrupted mice phenocopy norrin and frizzled4 mutant mice, and TSPAN12 is a strong facilitator of Norrin/Frizzled4 signaling in cell-based assays. We have three objectives: i) We hypothesize that TSPAN12 is functionally required in retinal endothelial cells and that restricted TSPAN12 expression modulates the spatio-temporal pattern of pathway activation. This model predicts that conditional disruption of the tspan12 gene in endothelial cells, but not in other cell types, recapitulates the loss-of-function phenotypes, e.g., lack of intraretinal capillaries and impaired blood-retina barrier integrity. We will use the Cre-Lox system to test this model. ii) TSPAN12 is required only in Norrin-induced but not in Wnt-induced Frizzled4 signaling. This is an intriguing and unexplained feature of Norrin/Frizzled4 signaling. We hypothesize that Norrin cannot efficiently induce protein interactions in the Norrin receptor complex and therefore operates together with TSPAN12. In contrast, Wnts may induce the required protein interactions autonomously (e.g., Norrin and Wnts differ in their ability to bind receptor and co-receptor simultaneously). We will test this hypothesis by characterizing protein interactions in the Norrin receptor complex, by determining which interactions are promoted by Norrin, Wnt3a, and TSPAN12, and by determining the consequences of abolishing or forcing relevant protein interactions in cell-based reporter assays. iii) Several recent human genetic studies independently reported mutations in tspan12 in patients with familial exudative vitreoretinopathy. We will determine if the reported mutations impair the function of TSPAN12 in Norrin/Frizzled4 signaling. Building on our investigation of TSPAN12 protein interactions, we will ask if one or several of the mutations impair critical protein interactions of TSPAN12. We will also determine if one or several of the mutations impair the folding or trafficking of TSPAN12. Conclusion: Norrin/Frizzled4 signaling is under investigation as a target for therapeutic intervention. A refined mechanistic model of TSPAN12 function in Norrin/Frizzled4 signaling should aid efforts to target this pathway in neovascular diseases of the retina.